Metabolic selection of a homologous recombination-mediated gene loss protects Trypanosoma brucei from ROS production by glycosomal fumarate reductase
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100548
ISSN1083-351X
AutoresMarion Wargnies, Nicolas Plazolles, Robin Schenk, Oriana Villafraz, Jean‐William Dupuy, Marc Biran, Sabine Bachmaier, Hélène Baudouin, Christine Clayton, Michael Boshart, Frédéric Bringaud,
Tópico(s)Synthesis and Biological Evaluation
ResumoThe genome of trypanosomatids rearranges by using repeated sequences as platforms for amplification or deletion of genomic segments. These stochastic recombination events have a direct impact on gene dosage and foster the selection of adaptive traits in response to environmental pressure. We provide here such an example by showing that the phosphoenolpyruvate carboxykinase (PEPCK) gene knockout (Δpepck) leads to the selection of a deletion event between two tandemly arranged fumarate reductase (FRDg and FRDm2) genes to produce a chimeric FRDg-m2 gene in the Δpepck∗ cell line. FRDg is expressed in peroxisome-related organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is nonfunctional and cytosolic. Re-expression of FRDg significantly impaired growth of the Δpepck∗ cells, but FRD enzyme activity was not required for this negative effect. Instead, glycosomal localization as well as the covalent flavinylation motif of FRD is required to confer growth retardation and intracellular accumulation of reactive oxygen species (ROS). The data suggest that FRDg, similar to Escherichia coli FRD, can generate ROS in a flavin-dependent process by transfer of electrons from NADH to molecular oxygen instead of fumarate when the latter is unavailable, as in the Δpepck background. Hence, growth retardation is interpreted as a consequence of increased production of ROS, and rearrangement of the FRD locus liberates Δpepck∗ cells from this obstacle. Interestingly, intracellular production of ROS has been shown to be required to complete the parasitic cycle in the insect vector, suggesting that FRDg may play a role in this process. The genome of trypanosomatids rearranges by using repeated sequences as platforms for amplification or deletion of genomic segments. These stochastic recombination events have a direct impact on gene dosage and foster the selection of adaptive traits in response to environmental pressure. We provide here such an example by showing that the phosphoenolpyruvate carboxykinase (PEPCK) gene knockout (Δpepck) leads to the selection of a deletion event between two tandemly arranged fumarate reductase (FRDg and FRDm2) genes to produce a chimeric FRDg-m2 gene in the Δpepck∗ cell line. FRDg is expressed in peroxisome-related organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is nonfunctional and cytosolic. Re-expression of FRDg significantly impaired growth of the Δpepck∗ cells, but FRD enzyme activity was not required for this negative effect. Instead, glycosomal localization as well as the covalent flavinylation motif of FRD is required to confer growth retardation and intracellular accumulation of reactive oxygen species (ROS). The data suggest that FRDg, similar to Escherichia coli FRD, can generate ROS in a flavin-dependent process by transfer of electrons from NADH to molecular oxygen instead of fumarate when the latter is unavailable, as in the Δpepck background. Hence, growth retardation is interpreted as a consequence of increased production of ROS, and rearrangement of the FRD locus liberates Δpepck∗ cells from this obstacle. Interestingly, intracellular production of ROS has been shown to be required to complete the parasitic cycle in the insect vector, suggesting that FRDg may play a role in this process. Trypanosomatids, including the human infective Leishmania and Trypanosoma species, present several biological singularities in comparison with classical eukaryotic model organisms. For instance, genes are transcribed constitutively as part of long polycistronic units where the precursor mRNA molecules are matured by coupled trans-splicing and polyadenylation (1Clayton C.E. Gene expression in Kinetoplastids.Curr. Opin. Microbiol. 2016; 32: 46-51Crossref PubMed Scopus (97) Google Scholar). As a consequence, gene regulation occurs mostly at the posttranscriptional, translational, and posttranslational levels with no control at the level of transcription initiation. Changes in gene copy number can also modulate gene expression and are therefore seen when selective pressure is applied. They usually arise from homologous recombination events between repeated sequences and are particularly common in Leishmania spp (2Ubeda J.M. Raymond F. Mukherjee A. Plourde M. Gingras H. Roy G. Lapointe A. Leprohon P. Papadopoulou B. Corbeil J. Ouellette M. Genome-wide stochastic adaptive DNA amplification at direct and inverted DNA repeats in the parasite Leishmania.PLoS Biol. 2014; 12e1001868Crossref PubMed Scopus (92) Google Scholar). In Leishmania, small repetitive sequences are widespread throughout the genome and recombination events appear stochastically with a frequency in the order of 10−6/10−7 per cell generation. They result either in the production of extrachromosomal DNA sequences or in the deletion of the DNA fragment located between the two recombinogenic repeats. Under selection pressure, such as exposition to drugs, a subpopulation with an advantageous amplicon conferring drug resistance can emerge (3Garvey E.P. Santi D.V. Stable amplified DNA in drug-resistant Leishmania exists as extrachromosomal circles.Science. 1986; 233: 535-540Crossref PubMed Scopus (81) Google Scholar, 4Grondin K. Papadopoulou B. Ouellette M. Homologous recombination between direct repeat sequences yields P-glycoprotein containing amplicons in arsenite resistant Leishmania.Nucleic Acids Res. 1993; 21: 1895-1901Crossref PubMed Scopus (87) Google Scholar, 5Papadopoulou B. Roy G. Ouellette M. 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Modulation of gene expression in drug resistant Leishmania is associated with gene amplification, gene deletion and chromosome aneuploidy.Genome Biol. 2008; 9: R115Crossref PubMed Scopus (125) Google Scholar, 9Leprohon P. Legare D. Raymond F. Madore E. Hardiman G. Corbeil J. Ouellette M. Gene expression modulation is associated with gene amplification, supernumerary chromosomes and chromosome loss in antimony-resistant Leishmania infantum.Nucleic Acids Res. 2009; 37: 1387-1399Crossref PubMed Scopus (127) Google Scholar). The genome of Trypanosoma brucei also contains a large number of sequence repeats (773) potentially leading to 1848 genetic recombination events, some of them already experimentally validated (2Ubeda J.M. Raymond F. Mukherjee A. Plourde M. Gingras H. Roy G. Lapointe A. Leprohon P. Papadopoulou B. Corbeil J. Ouellette M. Genome-wide stochastic adaptive DNA amplification at direct and inverted DNA repeats in the parasite Leishmania.PLoS Biol. 2014; 12e1001868Crossref PubMed Scopus (92) Google Scholar). So far, no DNA amplification (except for changes in ploidy and in gene copy number) has been observed upon specific selection, suggesting that deletions are more common (10Wilson K. Berens R.L. Sifri C.D. Ullman B. Amplification of the inosinate dehydrogenase gene in Trypanosoma brucei gambiense due to an increase in chromosome copy number.J. Biol. Chem. 1994; 269: 28979-28987Abstract Full Text PDF PubMed Google Scholar, 11Graf F.E. Ludin P. Wenzler T. Kaiser M. Brun R. Pyana P.P. Buscher P. de Koning H.P. Horn D. Maser P. Aquaporin 2 mutations in Trypanosoma brucei gambiense field isolates correlate with decreased susceptibility to pentamidine and melarsoprol.PLoS Negl. Trop. Dis. 2013; 7: e2475Crossref PubMed Scopus (52) Google Scholar, 12Mulindwa J. Leiss K. Ibberson D. Kamanyi Marucha K. Helbig C. 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In the glucose-free environment of its insect host (tsetse fly), the parasite depends on proline for its metabolism (14Coustou V. Biran M. Breton M. Guegan F. Riviere L. Plazolles N. Nolan D. Barrett M.P. Franconi J.M. Bringaud F. Glucose-induced remodeling of intermediary and energy metabolism in procyclic Trypanosoma brucei.J. Biol. Chem. 2008; 283: 16342-16354Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 15Mantilla B.S. Marchese L. Casas-Sanchez A. Dyer N.A. Ejeh N. Biran M. Bringaud F. Lehane M.J. Acosta-Serrano A. Silber A.M. Proline metabolism is essential for Trypanosoma brucei brucei survival in the tsetse vector.PLoS Pathog. 2017; 13e1006158Crossref PubMed Scopus (67) Google Scholar) and needs to produce hexose phosphates through gluconeogenesis from proline-derived phosphoenolpyruvate (PEP) to feed essential pathways (16Allmann S. Morand P. Ebikeme C. Gales L. Biran M. Hubert J. Brennand A. Mazet M. Franconi J.M. Michels P.A. Portais J.C. Boshart M. Bringaud F. Cytosolic NADPH homeostasis in glucose-starved procyclic Trypanosoma brucei relies on malic enzyme and the pentose phosphate pathway fed by gluconeogenic flux.J. Biol. Chem. 2013; 288: 18494-18505Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Two phosphoenolpyruvate-producing enzymes, PEP carboxykinase (PEPCK, EC: 4.1.1.32, Tb927.2.4210) and pyruvate phosphate dikinase (PPDK, EC 2.7.9.1, Tb927.11.3120) have a redundant function for the essential gluconeogenesis from proline (17Wargnies M. Bertiaux E. Cahoreau E. Ziebart N. Crouzols A. Morand P. Biran M. Allmann S. Hubert J. Villafraz O. Millerioux Y. Plazolles N. Asencio C. Riviere L. Rotureau B. et al.Gluconeogenesis is essential for trypanosome development in the tsetse fly vector.PLoS Pathog. 2018; 14e1007502Crossref PubMed Scopus (17) Google Scholar). In glucose-rich conditions, PPDK and PEPCK work in the opposite direction to produce pyruvate and oxaloacetate, respectively, in addition to ATP. This pathway is also essential to maintain the glycosomal redox balance (18Deramchia K. Morand P. Biran M. Millerioux Y. Mazet M. Wargnies M. Franconi J.M. Bringaud F. Contribution of pyruvate phosphate dikinase in the maintenance of the glycosomal ATP/ADP balance in the Trypanosoma brucei procyclic form.J. Biol. Chem. 2014; 289: 17365-17378Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Glycosomes are peroxisome-related organelles using the same machinery for protein import and harbor the 6 or 7 first glycolytic steps (19Opperdoes F.R. Borst P. Spits H. Particle-bound enzymes in the bloodstream form of Trypanosoma brucei.Eur. J. Biochem. 1977; 76: 21-28Crossref PubMed Scopus (72) Google Scholar). Because of the impermeability of the glycosomal membrane to bulky metabolites, such as cofactors and nucleotides, ATP molecules consumed by the first glycolytic steps (steps 1 and 3 in Fig. 1) need to be regenerated in the glycosomes by PPDK and PEPCK (step 14 and 15) (18Deramchia K. Morand P. Biran M. Millerioux Y. Mazet M. Wargnies M. Franconi J.M. Bringaud F. Contribution of pyruvate phosphate dikinase in the maintenance of the glycosomal ATP/ADP balance in the Trypanosoma brucei procyclic form.J. Biol. Chem. 2014; 289: 17365-17378Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Similarly, NAD+ molecules consumed in the glycosomes during glycolysis (step 6) have to be regenerated within the organelle by the succinic fermentation pathway composed of PEPCK, malate dehydrogenase (EC: 1.1.1.37, Tb927.10.15410, step 16), fumarase (EC: 4.2.1.2, Tb927.10.15410, step 17), and NADH-dependent fumarate reductase (FRDg, EC: 1.3.1.6, Tb927.5.930, step 18) (20Ebikeme C. Hubert J. Biran M. Gouspillou G. Morand P. Plazolles N. Guegan F. Diolez P. Franconi J.M. Portais J.C. Bringaud F. Ablation of succinate production from glucose metabolism in the procyclic trypanosomes induces metabolic switches to the glycerol 3-phosphate/dihydroxyacetone phosphate shuttle and to proline metabolism.J. Biol. Chem. 2010; 285: 32312-32324Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Alternatively, the glycosomal redox balance can be maintained by the glycerol 3-phosphate (Gly3P)/dihydroxyacetone phosphate (DHAP) shuttle, as observed for the PEPCK null mutant (Δpepck) and illustrated in Figure 1B (20Ebikeme C. Hubert J. Biran M. Gouspillou G. Morand P. Plazolles N. Guegan F. Diolez P. Franconi J.M. Portais J.C. Bringaud F. Ablation of succinate production from glucose metabolism in the procyclic trypanosomes induces metabolic switches to the glycerol 3-phosphate/dihydroxyacetone phosphate shuttle and to proline metabolism.J. Biol. Chem. 2010; 285: 32312-32324Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The T. brucei genome contains three FRD genes. Two are tandemly arranged in chromosome 5: they encode the glycosomal isoform (FRDg) and a potential FRD isoform for which expression has not been detected so far in trypanosomes (FRDm2, Tb927.5.940). The third gene, located on chromosome 10, codes for the mitochondrial isoform (FRDm1, Tb927.10.3650, step 21) (21Besteiro S. Biran M. Biteau N. Coustou V. Baltz T. Canioni P. Bringaud F. Succinate secreted by Trypanosoma brucei is produced by a novel and unique glycosomal enzyme, NADH-dependent fumarate reductase.J. Biol. Chem. 2002; 277: 38001-38012Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 22Coustou V. Besteiro S. Riviere L. Biran M. Biteau N. Franconi J.M. Boshart M. Baltz T. Bringaud F. A mitochondrial NADH-dependent fumarate reductase involved in the production of succinate excreted by procyclic Trypanosoma brucei.J. Biol. Chem. 2005; 280: 16559-16570Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) (Fig. 2B). FRDg is a 120 kDa protein composed of three domains, a N-terminal ApbE (Alternative pyrimidine biosynthesis protein)-like flavin transferase domain (pfam: PF02424), a central FRD domain (superfamily: SSF56425), and a C-terminal cytochrome b5 reductase (Cytb5R) domain (superfamily: SSF63380) (21Besteiro S. Biran M. Biteau N. Coustou V. Baltz T. Canioni P. Bringaud F. Succinate secreted by Trypanosoma brucei is produced by a novel and unique glycosomal enzyme, NADH-dependent fumarate reductase.J. Biol. Chem. 2002; 277: 38001-38012Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). In addition, FRDg has a conserved flavinylation motif at its extreme N terminus, shown to be required for FRD activity in the related organism Leptomonas pyrrhocoris (23Serebryakova M.V. Bertsova Y.V. Sokolov S.S. Kolesnikov A.A. Baykov A.A. Bogachev A.V. Catalytically important flavin linked through a phosphoester bond in a eukaryotic fumarate reductase.Biochimie. 2018; 149: 34-40Crossref PubMed Scopus (8) Google Scholar). We report here that two independent PEPCK null mutant cell lines express a chimeric nonfunctional FRDg-m2 isoform resulting from homologous recombination within the FRDg/FRDm2 locus. The selective advantage provided by the loss of the FRDg gene in the context of the PEPCK null background depends on the glycosomal localization of FRDg and the presence of the N-terminal putative flavinylation site. We propose that the absence of metabolic flux through the glycosomal succinic fermentation pathway in PEPCK null mutants made the FAD/FMN cofactors of FRDg available to oxygen for production of reactive oxygen species (ROS) in the organelles. In order to study possible changes in gene expression of mutants missing key enzymes involved in the maintenance of the glycosomal redox and ATP/ADP balances, we have compared the total proteomes of the parental, Δppdk (24Coustou V. Besteiro S. Biran M. Diolez P. Bouchaud V. Voisin P. Michels P.A. Canioni P. Baltz T. Bringaud F. ATP generation in the Trypanosoma brucei procyclic form: Cytosolic substrate level phosphorylation is essential, but not oxidative phosphorylation.J. Biol. Chem. 2003; 278: 49625-49635Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), Δpepck (20Ebikeme C. Hubert J. Biran M. Gouspillou G. Morand P. Plazolles N. Guegan F. Diolez P. Franconi J.M. Portais J.C. Bringaud F. Ablation of succinate production from glucose metabolism in the procyclic trypanosomes induces metabolic switches to the glycerol 3-phosphate/dihydroxyacetone phosphate shuttle and to proline metabolism.J. Biol. Chem. 2010; 285: 32312-32324Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), Δppdk/Δpepck (18Deramchia K. Morand P. Biran M. Millerioux Y. Mazet M. Wargnies M. Franconi J.M. Bringaud F. Contribution of pyruvate phosphate dikinase in the maintenance of the glycosomal ATP/ADP balance in the Trypanosoma brucei procyclic form.J. Biol. Chem. 2014; 289: 17365-17378Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), and Δppdk/Δpepck/RNAiGPDH.i (".i" stands for tetracycline-induced) cell lines, by label-free quantitative mass spectrometry. The effectiveness of this approach was confirmed by the 41.7–54.0-fold reduction observed for the PPDK and/or PEPCK peptide counts in the four mutant cell lines analyzed, compared with the parental cell line (see Fig. 2A, for the parental and Δppdk/Δpepck/RNAiGPDH.i cell lines and the PXD020185 data set on the ProteomeXchange Consortium repository for the other cell lines). Similarly, the GPDH signal was strongly reduced (20.4-fold) in the Δppdk/Δpepck/RNAiGPDH.i mutant. This analysis also showed that expressions of FRDg and FRDm2 were 6.9-fold decreased and tenfold increased, respectively, in the Δppdk/Δpepck/RNAiGPDH.i cell line, while expression of FRDm1 was not affected (Fig. 2A). In contrast, expression of the three FRD isoforms remained unaffected in the three other mutant cell lines (PXD020185 data set on the ProteomeXchange Consortium). This FRD expression pattern was confirmed by western blotting using immune sera specific to FRDg (αFRDg) and FRDm2 (αFRDm2), in addition to the αFRD immune serum produced against the conserved FRDg central domain, which is 100% and 71% identical with FRDm2 and FRDm1, respectively (Fig. 2, B and C). The αFRD antibodies recognized two proteins in both the parental and Δppdk/Δpepck/RNAiGPDH.i cell lines, including the ∼130 kDa FRDm1 isoform (Fig. 2, D and E). As previously reported, the second isoform expressed in the parental cell line (∼120 kDa) was recognized by the αFRDg, while no signal corresponding to FRDm2 was detected using αFRDm2 (24Coustou V. Besteiro S. Biran M. Diolez P. Bouchaud V. Voisin P. Michels P.A. Canioni P. Baltz T. Bringaud F. ATP generation in the Trypanosoma brucei procyclic form: Cytosolic substrate level phosphorylation is essential, but not oxidative phosphorylation.J. Biol. Chem. 2003; 278: 49625-49635Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In contrast, the ∼115 kDa protein expressed in the Δppdk/Δpepck/RNAiGPDH.i cell line was recognized by αFRDm2, but not αFRDg. This suggests that the mutant cell line switched from FRDg to FRDm2 expression, although the apparent size of the detected FRDm2 isoform was higher than the theoretical one (∼115 versus 94.8 kDa). Coomassie staining, western blotting (Fig. 2E), and proteomic analyses (Fig. 2A) of purified glycosomal fractions confirmed the glycosomal localization of FRDg expressed in the parental cell line. In contrast, none of the FRD isoforms were detectable by western blot in the glycosomal fractions of the mutant cell line, which is consistent with the proteomic analyses. To determine whether the mutually exclusive expression of FRDg and FRDm2 was related to genomic rearrangement inside the FRDg/FRDm2 locus, a Southern blot analysis was conducted using as probe the conserved FRDg/FRDm2 central domain, which hybridizes with the FRDm1 gene ("1" in Fig. 3A) but gives a much stronger signal for the FRDg and FRDm2 genes ("g" and "2" in Fig. 3A). The restriction pattern obtained with the NcoI-, PvuII-, NdeI-, and XhoI-digested parental genomic DNA (Fig. 3A) was consistent with the restriction map of the FRDm1 (Fig. 3B) and FRDg/FRDm2 (Fig. 3C) loci deduced from the T. brucei TriTrypDB database (strain 927). Although the FRDm1 locus was identical in the Δppdk/Δpepck/RNAiGPDH genome, the pattern observed for the FRDg/FRDm2 locus differed markedly (Fig. 3A). For instance, the 6.4 kb PvuII-fragment containing the two FRD genes in the parental genome was converted into a 2.3 kb PvuII-fragment in the mutant genome, suggesting that 4.1 kb had been deleted from the FRDg/FRDm2 locus. Analysis of the three other restriction profiles led to the same conclusion. The size of the deleted DNA fragment (4.1 kb) was consistent with the size of the theoretical DNA fragment (4074 bp) resulting from homologous recombination between the central 1450 bp FRD domains, which are 100% identical in the FRDg and FRDm2 genes. In conclusion, these data showed that a recombination event occurred between the FRDg and FRDm2 genes in the Δppdk/Δpepck/RNAiGPDH mutant to generate a FRDg-m2 chimeric gene coding for a FRD chimeric protein slightly smaller than FRDg (theoretical molecular weights: 120.6 versus 123.5 kDa, respectively). This DNA rearrangement event was present on both alleles of the locus, since the wild-type FRDg and FRDm2-amplified DNA fragments (Fig. 3C) and the endogenous FRDg protein (Fig. 2D) were not detectable in the mutant cell line. To further study this DNA rearrangement event, we used PCR with primer pairs designed for amplification of the central FRD domain of the FRDg (g5 and g3 primers), FRDm2 (m5 and m3 primers), and FRDg-m2 (g5 and m3 primers) genes (see Fig. 4A). As expected, the FRDg- and FRDm2-specific DNA fragments were amplified from the parental EATRO1125.T7T cell line but not from the Δppdk/Δpepck/RNAiGPDH genomic DNA (Fig. 4, B and C), confirming the loss of the wild-type FRDg/FRDm2 locus in the mutant cell population. Also in agreement with the Southern blot data, the FRDg-m2-specific fragment was amplified from the mutant genomic DNA. Interestingly, however, the FRDg-m2-specific fragment was also very weakly PCR-amplified from the parental EATRO1125.T7T cell line, which suggests that the recombination event stochastically occurred in the wild-type cells (Fig. 4, B and C). Moreover, a 1.5 kb PCR product was obtained using parental DNA and the g3 and m5 primers; we suggest that the template was a circularized version of the deleted fragment (Fig. 4, A–C). No corresponding PCR product was detected in the mutant cell line, suggesting that the circularized deleted DNA fragment was not replicated and thus diluted during cell division to become undetectable. The same PCR analysis conducted on genomic DNA samples showed that the rearrangement event occurred in other strains of T. brucei (Trypanosoma equiperdum, T. b. brucei, and T. b. rhodesiense) (Fig. 4B). We took advantage of the Δppdk/Δpepck/RNAiGPDH cell line being homozygous for the FRDg-m2 recombinant locus to calculate the allele frequency of FRDg-m2 in the EATRO1125.T7T parental cell line. We compared the FRDg-m2 copy number in the two lines by semiquantitative PCR using different amounts of genomic DNA and the g5-m3 primer pair. Primers specific for a control gene (fructose-1,6-bisphosphatase, Tb927.9.8720) were used for normalization (Fig. 5A). The results showed that the FRDg-m2 gene copy number is 3700-times higher in the Δppdk/Δpepck/RNAiGPDH homogeneous cell line relative to the heterogeneous parental population (Fig. 5B), indicating that at the time of analysis one in 1850 cells in the parental population had a hemizygous recombined allele. This estimation is at best an upper limit since we cannot exclude PCR artifacts due to premature termination of the PCR product in the conserved region of one isoform followed by priming on the other isoform. Altogether, this analysis suggested that the generation of the FRDg-m2 chimeric gene occurs at low frequency by homologous recombination in the T. brucei genome, but was specifically selected in the Δppdk/Δpepck/RNAiGPDH cell line. Analysis of the Δpepck mutant obtained and frozen in 2008 (20Ebikeme C. Hubert J. Biran M. Gouspillou G. Morand P. Plazolles N. Guegan F. Diolez P. Franconi J.M. Portais J.C. Bringaud F. Ablation of succinate production from glucose metabolism in the procyclic trypanosomes induces metabolic switches to the glycerol 3-phosphate/dihydroxyacetone phosphate shuttle and to proline metabolism.J. Biol. Chem. 2010; 285: 32312-32324Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), then thawed in 2013, and maintained for weeks in culture (see Fig. 4E, here named Δpepck∗), yielded more information about selection of the homologous recombination event in the FRDg/FRDm2 locus. It is noteworthy that this Δppdk/Δpepck/RNAiGPDH cell line was not derived from the Δpepck cell line (see Fig. 4E). Indeed, the Δpepck and the parental cells showed the same PCR profile, while the Δpepck∗ cells maintained for a long term in in vitro culture showed a pattern similar to the Δppdk/Δpepck/RNAiGPDH cell line. Therefore, selection of the recombinant allele occurred independently in a second mutant cell line (Fig. 4C). However, the selection process was probably less stringent or more recent in the PEPCK null background compared with the Δppdk/Δpepck/RNAiGPDH background, as illustrated by the presence of the wild-type locus in the Δpepck∗ population (g5-g3 and m5-m3 primer pairs in Fig. 4C), even after months of growth. As expected from the PCR analysis, the FRDg-m2 chimeric isoform was expressed in the Δpepck∗ cell line, but not detectable by western blotting in the Δpepck cells (Fig. 4D). It is noteworthy that proteomics analysis performed on the Δpepck cell line before long-term cultivation showed an intermediate profile of FRD isoform expression between the parental and the Δppdk/Δpepck/RNAiGPDH cell lines (PXD020185 data set on the ProteomeXchange Consortium). These data strongly suggest selection for the FRDg-m2 recombinant locus when PEPCK is missing (Fig. 4E). The glycosomal localization of FRDg was confirmed by a digitonin cell fractionation experiment. Western blot analysis of the supernatant fractions confirmed that as expected, the FRDg isoform was released together with the PPDK and PEPCK glycosomal markers (Fig. 6A). In contrast, the FRDg-m2 chimeric isoform expressed in the Δpepck∗ cell line was released at lower digitonin concentrations (0.03 mg versus 0.14 mg of digitonin per mg of protein) together with the enolase cytosolic marker (Fig. 6A). The cytosolic location of the FRDg-m2 chimeric isoform expressed in the Δpepck∗ cell line was confirmed by a western blot analysis of glycosomal and cytosolic fractions prepared by differential centrifugation after silicon carbide cell homogenization (Fig. 6B). The NADH-dependent FRD (NADH-FRD) activity was determined in the glycosomal and cytosolic fractions of the original EATRO1125.T7T and the Δpepck∗ cell lines. As expected, NADH-FRD activity was detected in the glycosomal fraction of cells expressing FRDg (EATRO1125.T7T), but not in the glycosomes of the Δpepck∗ cell line (Fig. 6B). The low level of NADH-FRD activity detected in the cytosolic fraction of the EATRO1125.T7T cell line, compared with the glycosomal fraction (2.3%), was presumably due to the lysis of a few glycosomes during the grinding step. The absence of NADH-FRD activity in the cytosolic fraction of the Δpepck∗ cell line demonstrates that the chimeric FRDg-m2 isoform is inactive (Fig. 6B). These data highlight the role of the Cytb5R domain in the NADH-FRD activity, since only this domain differs between the active FRDg and inactive FRDg-m2 isoforms. Selection of the FRDg-m2 recombinant locus in the Δpepck∗ and Δppdk/Δpepck/RNAiGPDH cell lines implied that either expression of the FRDg-m2 isoform in the cytosol or abolition of FRDg expression in the glycosomes provided a selective advantage to both mutant cell lines. To determine which of these two hypotheses is correct, tetracycline-inducible ectopic expression of FRDg and RNAi-mediated downregulation of FRDg-m2 were performed in the Δpepck∗ cell line (Δpepck∗/OEFRDg and Δpepck∗/RNAiFRDg-m2, respectively) (Fig. 7A). These experiments could not be conducted with the Δppdk/Δpepck/RNAiGPDH cell line, because all five available selectable markers had already been used. The glycosomal localization of the recombinant FRDg in the Δpepck∗/OEFRDg.i line was confirmed by western blotting and enzymatic activity assay of glycosomal fractions (Fig. 6B). The doubling time of the Δpepck∗/RNAiFRDg-m2 cell population was identical in the absence (.ni) or the presence (.i) of tetracycline, indicating that expression of the FRDg-m2 chimera was well tolerated by the Δpepck∗ mutant. In contrast, induction of FR
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